Heparan sulfate (HS) is an essential glycan that is present in large quantities on the cell surface and in the extracellular matrix. HS participates in a variety of physiological and pathophysiological functions, including blood coagulation, inflammatory response and cell differentiation. HS is a highly sulfated polysaccharide. Heparin, a special form of HS, is a commonly used anticoagulant drug. The wide range of biological functions of HS attracts considerable interest in exploiting heparin or heparin-like molecules for the development of anticancer and antiviral drugs. The uniquely distributed sulfation pattern of HS is believed to regulate its functional specificity. However, chemical synthesis of HS, especially those larger than hexasaccharides, is extremely difficult. Using HS biosynthetic enzymes, our labs can produce an array of HS with unique sulfation patterns and functions. Our success has proved the feasibility of conducting enzyme-based synthesis of HS with unique biological activities. The long term aim of this project is to develop a method to synthesize HS with high structural precision. Our hypothesis is that the distribution of N- sulfoglucosamine residues determines the susceptibility of all subsequent modifications during heparan sulfate biosynthesis, including epimerization, 2-O-sulfation, 6-O- sulfation, and 3-O-sulfation. In this proposal, we will synthesize structurally defined oligosaccharides carrying N-sulfoglucosamine residues using glycosyl transferases. We will then determine the substrate specificities of a variety of HS biosynthetic enzymes with the aim of precisely positioning the 6-O-sulfo and 3-O-sulfo groups as well as 2-O- sulfo iduronic acid within oligosaccharides. Finally, we plan to utilize this method to investigate novel anticoagulant HS structures. The success of this project will lead a comprehensive new approach to investigate the structure and function relationship of HS, potentially leading to the development of novel HS-based therapeutic reagents. Public Health Relevance: Heparan sulfate (HS) is a highly sulfated polysaccharide. Heparin, a special form of HS, is a commonly used anticoagulant drug. The wide range of biological functions of HS attracts considerable interest in exploiting heparin or heparin-like molecules for the development of anticancer and antiviral drugs. The uniquely distributed sulfation pattern of HS is believed to regulate its functional specificity. The long term aim of this project is to develop a method to synthesize HS with high structural precision. The success of this project will lead a comprehensive new approach to investigate the structure and function relationship of HS, potentially leading to the development of novel HS-based therapeutic reagents.